Abstract

Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multi-ubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substrate-linked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.

Fig. 1. Sequence similarity between UBA domains in Rad23 and certain Ub-isopeptidases (identical residues are indicated in bold). The numbers on the right indicate the amino acid residue at the C-terminus of the conserved UBA sequence in each respective protein. The upper two lines represent the sequence of the two UBA domains in yeast Rad23 protein. Only UBA1 sequences from human and mouse Rad23 are shown.

Fig. 2. Rad23 prevents the passage of Ub (∼8500 mol. wt) through a 30 000 mol. wt cut-off filter. (A) A Coomassie Blue stained gel showing the amounts of Rad23 and BSA (µg) that were used in a Ub-binding assay using Centricon-30. (B) Rad23 and BSA were added to ubiquitylation reactions that contained [32P]Ub, and subjected to ultrafiltration in Centricon-30 (autoradiogram). The retentate was separated on a 12% polyacrylamide gel and exposed to X-ray film. The retention of [32P]Ub increased in the presence of higher levels of Rad23 (lanes 1–3), while only a trace amount of [32P]Ub was recovered in reactions that contained BSA (lanes 4–6).

Fig. 3. Direct interaction between Rad23 and Ub. (A) GST, GST–Rad23 and GST–ΔUbLrad23 were purified to homogeneity from yeast, and equal amounts of protein (0.1 µg) were adsorbed to glutathione–Sepharose (lanes 1–3). The adsorbed proteins were incubated with unlabelled Ub (2.5 µg), and the affinity beads were washed and suspended in SDS–electrophoresis buffer. Proteins were separated by SDS–PAGE, transferred to nitrocellulose, and the upper half of the filter was incubated with anti-GST antibodies. Arrows to the right indicate the positions of GST–Rad23 (∼85 000 mol. wt) and GST–ΔUbLrad23 (∼75 000 mol. wt). The higher molecular weight species in lanes 2 and 3 are believed to be ubiquitin-conjugated derivatives of the GST fusion proteins. GST (∼26 000 mol. wt) is not visible. (B) The lower half of the nitrocellulose filter from (A) was incubated with antibodies against Ub. We detected an interaction between Ub and both GST–Rad23 and GST–ΔUbLrad23 (lanes 2 and 3), but not with GST (lane 1). Lane 4 contained 25 ng of purified Ub. (C) The UBA domains in Rad23 were expressed as fusions to GST (GST–UBA1 and GST–UBA2) and purified from E. coli. Equal amounts of the purified proteins were adsorbed to glutathione–Sepharose and incubated in binding buffer containing unlabelled Ub. A control reaction contained only GST protein (lane 1). The affinity beads were washed in buffer containing 0.5% Triton X-100 and the bound proteins examined in an immunoblot. We found that GST–UBA1 formed a strong interaction with Ub and di-Ub (lane 2), while GST–UBA2 formed a weaker interaction. Lane 4 contains a sample of the commercial Ub used in this study, and the positions of Ub and di-Ub are indicated. (D) Extracts that were prepared from a yeast strain that expressed Flag-Rad23 and GST–Ub were applied to glutathione–Sepharose beads. Following incubation at 4°C, the beads were washed extensively in buffer containing 0.5% Triton X-100, suspended in SDS-containing gel-loading buffer and resolved by SDS–PAGE. The separated proteins were transferred to a nitrocellulose filter and incubated with antibodies against the Flag epitope. Lane 1 contains an aliquot of the cell extract, and the arrow on the right indicates the position of Flag-Rad23 (∼60 000 mol. wt). Flag-Rad23 was precipitated only from extracts that contained GST–Ub (lane 3), but not if the strain did not express GST–Ub (lane 2). The bracket to the right indicates the presence of high molecular weight derivatives of Flag-Rad23, which could represent conjugation to Ub (also see A). (E) In a reciprocal experiment, we incubated yeast cell extracts with antibodies against the Flag epitope and examined the precipitated proteins for the presence of GST–Ub. Lane 1 shows the position of GST–Ub in total extracts. A low amount of GST–Ub (∼35 000 mol. wt) was non-specifically precipitated from an extract that lacked Flag-Rad23 (lane 2), although higher amounts were detected if the extract contained Flag-Rad23. An asterisk indicates an antibody cross-reaction against the IgG light chain (∼22 000 mol. wt).

Fig. 4. The UBA domains in Rad23 are required for efficient inhibition of multi-Ub chain assembly. (A) Substrate-linked multi-Ub chains were assembled efficiently in a ubiquitylation reaction that lacked Rad23 protein (see bracket adjacent to lane 1). However, in the presence of Rad23 the assembly of multi-Ub chains on H2B was significantly reduced (lane 2). As we reported previously, Rad23 itself becomes mono-ubiquitylated in these reactions (arrow adjacent to lane 2). (B) Lanes 3–5 represent ubiquitylation reactions that contained rad23ΔUBA1, rad23ΔUBA2 and rad23ΔUBA1,2. Rad23 single mutants, lacking either UBA1 (lane 3) or UBA2 (lane 4), could efficiently inhibit the formation of multi-Ub chains. In contrast, a Rad23 mutant that lacked both UBA domains (lane 5) was a poor inhibitor of substrate-linked multi-Ub chain assembly (see bracket adjacent to lane 5). Similar to the mono-ubiquitylation of Rad23, all three UBA mutants (lanes 3–5) were also ligated to a single [32P]Ub in these reactions (arrows adjacent to lanes 3 and 5). (A) and (B) represent the same experiment and gel, but the exposure of (B) was 4-fold longer to illustrate the defect in multi-Ub chain inhibition by rad23ΔUBA1,2.